Experiment 8.The Drug Release in Vitro and Percutaneous Penetration of Salicylic Acid

Experiment 8.The Drug Release In Vitro and Percutaneous Penetration of Salicylic Acid In Ointment

Li Yanzhi(0641628)

【Abstract】

Purposes:To be familiarwith the influence of the bases of ointments on the drug release; To learn to measure the drug releaserate and other parameters in the ointments using the method of percutaneous penetration; To know the preparation of the skin used in the experiment.

Methods: Percutaneous penetration, as a classicalmethod in vitro to study the drug release in the ointments, is employed to calculate the Q(permested cumulative amount per unit area) and the resulting J(permeability coefficient).

Results: We obtained, respectively, permested cumulative amount per unit area and the permeability coefficient of four kind of bases of ointments.

Conclusions: Bases of ointments influence the release of the drug in ointments through the transdermal.

【Keyword】ointment , base , percutaneous penetration, Franz diffusion cells

The method of percutaneous penetration is a classical method in vitro to study the drug release in the ointments. This method includes placing the ointments on the surface of the hairless skin in vitro, setting the skin into Franz diffusion cells and measuring the permeated cumulative amount of drug on the steady state of this system. At last you can compare the rates and the lag times of percutaneous penetration to analyze the influence of bases on the percutaneous penetration of a drug.

Generally, the percutaneous penetration of a drug is procedure of passive diffusion. In this experiment, we use hairless skin as a simple membrane. On the condition of keeping the concentration gradient of the two sides of the membrane, when a steady state arrives in the procedure of percutaneous penetration, the relationship of Q (the permeated cumulative amount per unit area) with t (the time of diffusion) is as follows:

Note: k -- the permeated coefficient.

On a steady state of the system, you can have a line when plot the permeated cumulative amount per unit area as a function of the diffusion time, and k is the slope of the line, while the lag time (Tl) is the x-intercept of the line.

【Equipments and materials】

1.Equipments

721-type UV spectrophotometer, water bath with constant temperature controlled by electric, magnetic force whisk, improved Franz diffusion cells, filter membrane (Φ25mm×0.45μm), test tubes (10ml), injectors (5ml), pipet (1ml), scraper, operating scissors, surgical clamps, evaporating dish

2.Materials

Salicylic acid, 6% Na2S (depilatory), ammonium ferric sulfate color developing reagent, stearic acid, glycerol monostearate, liquid paraffin, white Vaseline, wool fat, triethanolamine, tween 80, beeswax, paraffin, span 80, calcium hydroxide, CMC-Na, Mice (×5, 18~22g/each)

【Procedures】

1.Formulation and preparations

Refer to Exp.7

2.Preparation of hairless skin of mouse in vitro

1)Mice are killed by neck dissolution.

2)Clip the ventral hair off, wipe off the hair with 6%Na2S.

3)3 min later wash the skin with physiological saline solution.Peel off the skin.

4)Remove the fat tissue and wash the skin with physiological saline solution, then the hairless skin of mice can be got.

3.Preparation of a standard curve

1)Reference substance (salicylic acid) 125.00mg, precisely, into 250ml volumetric flask, and add proper volume with water.

2)Withdraw 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0ml of the above solution, place in 25ml volumetric flask, dilute to volumetric with water.

3)Withdraw 5ml of the solution and place in test tube, and add 1ml ammonium ferric sulfate color developing reagent and mix fully.

4)Measure the absorbance at 530nm by employing the 721 ultraviolet spectrophotometer.

4.The introduce of improved Franz diffusion cells

The diffusion area is 2.00 cm2. The diffusion cells are placed in a thermostatic water bath with a constant temperature of 37±1℃. The receptor compartment is stirred with a magnetic stir bar to keep a dynamic condition.

5.Experimental operation

1)Fix the above skin between the two compartments and add 2g ointment into the diffusion compartment.

2)Fill the receptor compartment with physiological saline solution, and note the volume（V）and stir continuously.

3)Withdraw a sample of 5ml of the solution at 1.0, 1.5, 2.0, 2.5, 3.0 hours, respectively, and filter through 0.45μm membrane filter and supply 5ml of physiological saline solution immediately to maintain the volume constant.

4)Measure accurately 2.5ml of the filtrate solution into 10ml test tube, and add 0.5ml of the color developing reagent and mix fully.

5)Measure accurately 2.5ml of the solution marinated with blank skin, and add 0.5ml color developing reagent and mix fully, which is as blank.

6)Measure the absorbance (A) of the sampling solutions at 530nm using a spectrophotometer. According to the standard curve, concentrations of salicylic acid can be got.

【Data processing and experimental Results】

1.The standard curve

Table1.Data of calibration curve of salicylic acid
V(ml) / C(ug/ml) / A
1.0 / 16.67 / 0.098
2.0 / 33.33 / 0.19
3.0 / 50.00 / 0.286
4.0 / 66.67 / 0.376
5.0 / 83.33 / 0.475
6.0 / 100.0 / 0.579

Equation:C = 174.04A + 0.2048(ug/ml)

2.Calculate the permeated cumulative amount and establish the curve of percutaneous penetration

Diffusion area S=2.00cm2

(Q=ug/cm2)

Cn: the drug concentration (ug/ml) of the point of n

Ci: the drug concentration (ug/ml) of the point of i

V: the total volume of the receptor compartment

Vi: the sample volume every time

S: the diffusion area

Establish a percutaneous penetration curve of salicylic acid by plotting the permeated cumulative amount per unit area with the time of diffusion. The lag time (T) is the x-intercept of the line extended from the linear part of the curve tail.

1)Group 1. O/W emulsion base

Table2-1 O/W emulsion base
Time(h) / △A / V1(ml) / A1 / A / C′(ug/ml) / C(ug/ml) / Q(ug/cm2)
1.0 / 0.000 / 16.00 / 0.010 / 0.010 / 1.945 / 2.334 / 18.67
1.5 / 0.091 / 0.091 / 16.04 / 19.25 / 159.8
2.0 / 0.121 / 0.121 / 21.26 / 25.52 / 258.1
2.5 / 0.176 / 0.176 / 30.84 / 37.00 / 413.8
3.0 / 0.189 / 0.189 / 33.10 / 39.72 / 528.0
Time(h) / V2(ml) / A2 / A / C′(ug/ml) / C(ug/ml) / Q(ug/cm2)
1.0 / 18.00 / 0.050 / 0.050 / 8.907 / 10.69 / 96.19
1.5 / 0.133 / 0.133 / 23.35 / 28.02 / 278.9
2.0 / 0.182 / 0.182 / 31.88 / 38.26 / 441.1
2.5 / 0.226 / 0.226 / 39.54 / 47.45 / 619.4
3.0 / 0.230 / 0.230 / 40.23 / 48.28 / 745.6
Time(h) / V3(ml) / A3 / A / C′(ug/ml) / C(ug/ml) / Q(ug/cm2)
1.0 / 16.00 / 0.075 / 0.075 / 13.26 / 15.91 / 127.3
1.5 / 0.141 / 0.141 / 24.74 / 29.69 / 277.3
2.0 / 0.201 / 0.201 / 35.19 / 42.22 / 451.8
2.5 / 0.256 / 0.256 / 44.76 / 53.71 / 649.3
3.0 / 0.252 / 0.252 / 44.06 / 52.88 / 776.8
Time(h) / Q1(ug/cm2) / Q2(ug/cm2) / Q3(ug/cm2) / Q(ug/cm2)
1 / 18.67 / 96.19 / 127.3 / 80.71
1.5 / 159.8 / 278.9 / 277.3 / 238.7
2 / 258.1 / 441.1 / 451.8 / 383.7
2.5 / 413.8 / 619.4 / 649.3 / 560.8
3 / 528.0 / 745.6 / 776.8 / 683.5

2)Group 2. W/O emulsion base

Table2-2 W/O emulsion base
Time(h) / V(ml) / △A / A1 / A2 / A3 / A / C(ug/ml) / Q(ug/cm2)
1.0 / 18.70 / 0.005 / 0.025 / 0.020 / 0.005 / 0.012 / 2.682 / 25.08
1.5 / 0.120 / 0.032 / 0.052 / 0.063 / 13.40 / 125.3
2.0 / 0.125 / 0.120 / 0.085 / 0.105 / 22.17 / 207.3
2.5 / 0.140 / 0.100 / 0.103 / 0.109 / 23.08 / 215.8
3.0 / 0.120 / 0.050 / 0.090 / 0.082 / 17.30 / 161.8

3)Group 3. water soluble base

Table2-3 water soluble base
Time(h) / V(ml) / △A / A1 / A2 / A3 / A / C(ug/ml) / Q(ug/cm2)
1.0 / 15.00 / 0.015 / 0.070 / 0.095 / 0.161 / 0.094 / 19.81 / 148.6
1.5 / 0.050 / 0.095 / 0.075 / 0.058 / 12.43 / 142.7
2.0 / 0.060 / 0.100 / 0.145 / 0.087 / 18.35 / 218.2
2.5 / 0.185 / 0.100 / 0.140 / 0.127 / 26.70 / 326.7
3.0 / 0.150 / 0.205 / 0.125 / 0.145 / 30.53 / 422.2

4)Group 4. oleaginous base

Table2-4 oleaginous base
Time(h) / V(ml) / △A / A1 / A2 / A3 / A / C(ug/ml) / Q(ug/cm2)
1.0 / 18.70 / 0.005 / 0.011 / 0.016 / 0.006 / 0.006 / 1.499 / 14.01
1.5 / 0.015 / 0.010 / 0.016 / 0.009 / 2.056 / 22.97
2.0 / 0.016 / 0.018 / 0.012 / 2.752 / 34.62
2.5 / 0.023 / 0.025 / 0.019 / 4.214 / 55.17
3.0 / 0.022 / 0.028 / 0.019 / 0.018 / 4.005 / 63.75

5)Group 5. O/W emulsion base

Table2-5 O/W emulsion base
Time(h) / V(ml) / △A / A1 / A2 / A3 / A / C(ug/ml) / Q(ug/cm2)
1.0 / 18.70 / 0.005 / 0.030 / 0.080 / 0.291 / 0.129 / 27.12 / 253.5
1.5 / 0.032 / 0.066 / 0.312 / 0.132 / 27.74 / 327.2
2.0 / 0.060 / 0.150 / 0.285 / 0.160 / 33.66 / 451.9
2.5 / 0.102 / 0.250 / 0.349 / 0.229 / 48.00 / 670.1
3.0 / 0.115 / 0.270 / 0.335 / 0.235 / 49.33 / 802.5

6)Group 6. W/O emulsion base

Table2-6 W/O emulsion base
Time(h) / V(ml) / △A / A1 / A2(稀释一倍) / A2 / A3 / A / C(ug/ml) / Q(ug/cm2)
1.0 / 18.70 / -0.005 / 0.394 / 0.555 / 1.110 / 0.291 / 0.603 / 126.3 / 1180
1.5 / 0.250 / 0.302 / 0.604 / 0.312 / 0.394 / 82.46 / 1087
2.0 / 0.145 / 0.225 / 0.450 / 0.285 / 0.298 / 62.55 / 1107
2.5 / 0.237 / 0.177 / 0.354 / 0.349 / 0.318 / 66.73 / 1302
3.0 / 0.260 / 0.226 / 0.452 / 0.335 / 0.354 / 74.18 / 1539

3.The rate of percutaneous penetration

You can have a line when plot the permeated cumulative amount per unit area(Q) at the linear part of the curve tail of the percutaneous penetration as a function of the diffusion time(t) ang J(ug/cm2·h) is the slop of the line.

Table3 compare of the percutaneous penetration of four bases
kind of bases / Equation(Q-t) / J′(ug/cm2·h) / J(ug/cm2·h) / Lag,T′(h) / Lag,T(h)
O/W emulsion base / Q = 254.61t - 184.65 / 254.61 / 291.72 / 0.725226817 / 0.631658204
Q = 328.83t - 176.94 / 328.83 / 0.53808959
W/O emulsion base / Q = 192.54t - 178.79 / 192.54 / 312.22 / 0.928586268 / 0.928586268
Q = 431.9t + 236 / 431.9
water soluble base / Q = 157.81t - 123.85 / 157.81 / 157.81 / 0.784804512 / 0.784804512
oleaginous base / Q= 28.577t - 20.174 / 28.577 / 28.577 / 0.705952339 / 0.705952339

Through comparing the J, we find release rate of drug : W/O emulsion base〉O/W emulsion base〉water soluble base〉oleaginous base. This is irresponsive to the theory.

【Discussions】

1.基质占软膏的绝大部分，不仅起赋形剂的作用，也对软膏剂的质量有重要影响。基质在一定程度上影响药物的释放、穿透和吸收。由于实验条件的不同，得到的结果有时也不相一致。释药速度顺序依次为：水溶性基质〉O/W〉W/O〉油溶性基质。这主要与基质与水的亲和力有关。实验所得结果与理论并不一致。

2.Fick 第一定律表明药物扩散速度与皮肤厚度、温度、溶剂性质、药物性质、药物浓度等都有关系。如实验中脱毛过度或不足，均会使药物释放加快或减慢，影响实验结果。故应严格控制脱毛时间，脱毛后应立即用生理盐水冲洗干净，防止损伤角质层。又如实验温度应控制在体温37℃，高于或低于体温温度，影响药物释放速度，不能真实反映药物在人体的实际情况。

3.影响药物透皮吸收的因素：

1)生理因素。

皮肤的水合作用。水合作用使药物的渗透变得更容易。
角质层的厚度。实验选择的为小鼠腹部的皮肤。同一组软膏的扩散实验尽量使用相同部位的皮肤，因为皮肤部位的不同，角质层厚度不同，药物渗透与厚度有关。
皮肤条件。皮肤的病理条件会影响其渗透。
吸收面积。装上扩散池后在旋紧螺丝之前，应用镊子将皮肤拉紧，否则会增大扩散面积。

2）剂型因素与药物性质

药物剂量和药物浓度。
药物分子及脂溶性。
pH与pKa。
熔点与热力学活度。

4.接受室灌注生理盐水后，应将室内空气全部排除。磁力搅拌转速以能均匀混合液体为宜，转速太快会形成漩涡，减少扩散面积，转速太小接受室上、下层溶液难以混匀。

【conclusion】

Bases of ointments influence the release of the drug in ointments through the transdermal.